Selection of excipients for Ethosome Formulations of Imiquimod through Drug–excipient Compatibility testing for Skin Cancer: A Pre-Formulation Study

 

Bharti Choudhary*, Jitendra Banweer

School of Pharmaceutical Sciences,

Sanjeev Agrawal Global Educational University, Bhopal 462022, Madhya Pradesh, India.

*Corresponding Author E-mail: patelbharti90@gmail.com

 

ABSTRACT:

Skin cancer is a prevalent global issue, with traditional therapies often lacking drug penetration and bioavailability. Ethosomes, lipid-based vesicles, offer a promising solution for transdermal drug delivery, encapsulating both hydrophilic and lipophilic drugs and providing superior skin penetration. Imiquimod, an immune response modifier, is commonly used to treat non-melanoma skin cancers, but its effectiveness is hindered by poor skin penetration and local irritation. Ethosomal formulations offer an innovative approach to improve the bioavailability and therapeutic effect of Imiquimod by facilitating deeper skin penetration and sustained release. The development of ethosomes requires the careful selection of excipients that are compatible with both the drug and the delivery system. In this study, Phosphatidylcholine, Ethanol, and Propylene Glycol were chosen as excipients for the formulation of Imiquimod-loaded ethosomes. Phosphatidylcholine serves as the key phospholipid for vesicle formation, while Ethanol and Propylene Glycol act as solvents and penetration enhancers. Imiquimod's compatibility with excipients was investigated utilizing methods such as DSC, FTIR, XRD, and UV to identify any possible interactions. The results confirmed that Imiquimod is compatible with Phosphatidylcholine, Ethanol, and Propylene Glycol, with no significant chemical interactions. This indicates the suitability of these excipients for the future development of Imiquimod-loaded ethosomal formulations for the treatment of skin cancer, ensuring both stability and enhanced therapeutic efficacy.

 

KEYWORDS: Excipients, Ethosome, Imiquimod, Pre-Formulation.

 

 

INTRODUCTION:

The stratum corneum layer of the skin is the primary obstacle to transdermal administration, a likely method for both topical and systemic medication absorption.1 One of the primary methods to enhance treatment is the creation of a unique drug carrier system at the nanoscale.2 Ethosomes, malleable, non-invasive carriers with dynamic physicochemical properties, were developed by Touitou and colleagues and have a lot of promise for topical drug delivery and pharmaceutical technology.2,3

 

Ethosomes, deformable vesicles that contain a lot of alcohol, boost cell membrane lipid fluidity, improve drug penetration through stratum corneum, and entrap hydrophilic, lipophilic, and amphiphilic medicines.4,3 Basal cell carcinoma (BCC) is the most prevalent skin cancer among Caucasians, causing approximately four million annual cases and 95% of non-melanoma skin cancers in the US.5 Due to ethanol, ethosomes have flexible and less tightly packed phospholipid bilayer membranes and better skin permeability than liposomes. Enhanced drug delivery via ethosomes is due to vesicle pliability, ethanol fluidisation, smaller vesicle diameter, and facilitated interaction with stratum corneum components. Ethanol enhances skin permeation by disrupting lipid organisation in the stratum corneum, extracting lipidic layers, and reducing lipid density and impermeability to permeants. Using ethanol to fluidise lipid bilayers in the stratum corneum allows small, flexible ethosomes to reach deeper skin layers. Unlike deformable liposomes, ethosomes improve medication penetration by breaking the skin's lipophilic barrier. Research has shown that ethosomes can carry their payload through the skin more effectively than traditional liposomes, which tend to stay in the upper layers.6 Imiquimod, a non-surgical treatment for BCC, stimulates both innate and acquired immune systems by triggering pro-inflammatory cytokines, resulting in maximum skin restoration.7,8 Research conducted on grown tumor cells has also demonstrated that imiquimod can directly promote apoptosis at high dosages.9 The FDA has approved topical formulations, such as AldaraTM (5% w/w imiquimod) cream, to treat superficial BCC.10 Single 250 mg sachets of AldaraTM cream, containing 12.5 mg of imiquimod, are available.11 The booklet with patient information that provides instructions to the user to cover a treatment area that is no larger than 20 cm2 with a thin layer of cream.12 Because the cream is provided as an outpatient service, the patient can apply the treatment to the afflicted skin areas at home. Despite the manufacturer's suggested dosage, the patient will determine how much cream is really administered per unit area of skin in a 250 mg sachet, and research has shown that it may be improperly applied over a significantly wider area of 386 cm2.13 When imiquimod is self-administered, dosage differences may cause variances in clinical response. A continuous delivery system with occlusion against water and fluids may be advantageous. Topical use of anticancer drugs may be an additional therapeutic option for nonmelanoma skin cancer.14 Sincere efforts have been made in this study to highlight the ethosomes' qualities, characteristics, evaluation criteria, methods, and patents to date.

 

MATERIAL AND METHODS:

During pre-formulation experiments, a UV-Visible spectrophotometer was used to determine the drug's compatibility with imiquimod.

 

Materials:

Teva API India Ltd provided Imiquimod API, while Fine Chem. Pvt. Ltd supplied Soya Phosphatidyl Choline, Propylene glycol, and Carbopol 934, while ethanol was procured from Shri Maruti Chem Enterprise Private Limited.

 

Physical properties:

·      Melting Point:

To ascertain imiquimod's melting point and verify its purity, a minimal amount of the medication was taken and measured three times.

·      Partition coefficient:

The study determined the partition coefficient of imiquimod, indicating its hydrophobic nature, using an octane-water system and separating funnel method, revealing two layers.

·      Solubility Study

The study analyzed Imiquimod's solubility in various solvents and phospholipids for Imiquimod Ethosomal gel formulation, revealing its maximum solubility in Lactic acid and Phosphatidyl Choline.

·      FTIR Spectrophotometry:

The study used solid sampling technique to analyze imiquimod's structure, preparing a sample of 1 mg drug and 100 mg dehydrated spectroscopic rank KBr, and comparing peaks to known functional group frequencies.

·      X-ray diffraction analysis (XRD):         

A popular diagnostic method for evaluating resources, X-ray diffraction (XRD) helps classify materials by exposing concentric rings of scattering peaks in the crystal lattice.

·      Differential scanning calorimetry (DSC):

pure drug thermogram of Imiquimod revealed a significant reduction in the melting peak of crystalline Imiquimod, indicating the formation of an amorphous phase when mixed with amorphous phosphatidylcholine.

·      Determination of maximum absorption (λ-max in 0.1N HCl) in UV Spectrophotometer

Preparation of 0.1N HCl:

To make 0.1N HCl, 1000 milliliters of pure water were mixed with 8.5 milliliters of strong hydrochloric acid. Samples were mixed with 0.1N HCl to create varying concentrations of Imiquimod. UV-visible spectrophotometer was used to measure absorbance, and a standard curve was created.

 

RESULT AND DISCUSSION:

·       Melting point:

Imiquimod’s melting point was discovered to be 291℃, which is comparable to the value provided and indicates sample purity.

·       Partition coefficient:

Partition coefficient study was performed by using shake flask method. The LogP of imiquimod is found to be 2.05±0.23 which indicates hydrophobic nature of drug.

·       Solubility study:

From the Above data (Table-1 and Table-2) it was found that Imiquimod has maximum solubility in Lactic acid (Solvent) and Phosphatidyl Choline (Phospholipid).

 

Table 1: Solubility profile of Imiquimod in different Solvents

Solvent

Amt(mg/ml)

Solvent

Amt(mg/ml)

Methanol

0.697±0.002

DMF

8.125±0.003

Ethanol

0.219±0.006

PEG400

0.349±0.003

DMSO

0.851±0.006

Oleic Acid

09.212±0.013

Lactic acid

46.021±0.004

Water

0.019±0.022

 

Table 2: Solubility profile of Imiquimod in different Phospholipids

Lipid

Amt(mg/ml)

Cholesterol

0.543±0.0025

Phospholipoid 90G

0.5885±0.0179

Phosphatidyl Choline

0.967±0.0485

FTIR spectrophotometry:

 

 

Fig. 1: IR spectrum of Imiquimod API

Fig. 2: IR spectrum of Phosphatidylcholine

 

 

Fig. 3: IR spectrum of Propylene glycol

Fig. 4: IR spectrum of Carbopol 934

 

Fig. 5: IR spectrum of Physical mixture

 

X-Ray diffraction analysis (XRD):

The polymorphic form of API is analyzed using the XRD pattern of the pure medication imiquimod.

 

Peak Position 2Ѳ Value

Peak Area

Integral Breadth (FWHM)

Crystallite size

Average crystallite size (nm)

Area of crystalline peak

Total Area (Amorphous + Crystalline)

Percentage Crystallites

D

=

Kλ

βCOSѲ

Crystallite size / 14

60.34731

299.35335

0.659634615

-10.39580301

29.62877129

4255

5094.47865

83.53%

73.91617

962.73905

0.047822376

-86.47508063

81.9521

1164.8121

0.117456002

-49.94156843

68.51497

578.7039

0.072107955

36.5077482

39.86228

79.1756

0.10401938

58.85689774

40.71856

88.97565

0.061545159

92.08605312

21.35329

8.3282

0.036420339

217.5519961

56.18563

294.3426

0.072406564

-92.19533644

64.2994

327.27045

0.101598424

-75.02522856

50.53293

120.04165

0.102879082

38.58552726

26.9521

11.9971

0.1069

73.90823554

51.19162

256.04565

0.10935

-44.75067384

36.6.473

35.32785

0.065628667

115.3419348

35.58084

28.6065

0.110519167

69.6533456

 

Fig. 6: Imiquimod Crystallinity Labeled Peaks

 

Fig. 7: Imiquimod Percentage Crystallinity

Fig. 8: Imiquimod Average Crystallite size

 

Differential scanning calorimetry (DSC):

The melting point and purity of the material were ascertained by recording the thermogram of pure imiquimod. A Mettler TA 4000 device with a DSC 25 cell was used to perform the DSC analysis.14

 

Fig. 9: DSC thermogram of Imiquimod

Corresponding to the melting point of crystalline Imiquimod. This peak is typically sharp and well-defined temperature characteristic which indicates that drug is of crystalline form.

 

Fig. 10: DSC thermogram of Phosphatidylcholine

Phosphatidylcholine is not showing a sharp melting point and well-defined temperature characteristic which indicates that drug is of Amorphous form

 

Fig. 11: DSC thermogram of Physical Mixture (Drug + Phosphatidylcholine)

The sharp melting peak of Imiquimod may reduce in intensity which indicating a loss of crystalline structure and the formation of an amorphous phase

 

Determination of absorption maxima (λmax in 0.1N HCl):

 

Fig. 12: Scan spectra of Imiquimod in 0.1N HCl

 

Determination of Calibration Curve data of imiquimod in 0.1N HCl:

Concentration (µ/ml)

Absorbance 0.1N HCl at 243 nm

0

0

2

0.105

4

0.211

6

0.287

8

0.427

10

0.527

 

 

Method validation method for UV-Vis spectrophotometry:

Parameter

Results

Acceptance Criteria

Linearity and Range

2–20 µg/mL

R² ≥ 0.999

Accuracy

(% Recovery)

99.4–100.8%

98–102%

Precision (%RSD)

0.33–0.44%

≤2%

LOD

0.15 µg/mL

As low as possible

LOQ

0.44 µg/mL

As low as possible

Robustness (%RSD)

<1%

≤2%

Ruggedness

No significant variation

Consistent results

Specificity

No interference

No interference

 

CONCLUSION:

The pre-formulation study and drug-excipient compatibility evaluation of Imiquimod-loaded ethosomal formulations demonstrated favourable results, supporting the potential for this drug delivery system in skin cancer treatment. The study confirmed that Phosphatidylcholine, Ethanol, and Propylene Glycol are compatible with Imiquimod, as no significant chemical interactions were observed. DSC analysis showed no shifts in the melting point of Imiquimod when mixed with the excipients, indicating stable physical properties. FTIR spectroscopy revealed no new peaks or alterations in the spectra of Imiquimod, Phosphatidylcholine, Ethanol, or Propylene Glycol, further confirming the lack of interaction. Additionally, UV analysis showed that the drug retained its integrity in the presence of the excipients, with >98% of Imiquimod remaining intact after 30 days of stability testing. The successful compatibility study ensures that these excipients can be used in the formulation of Imiquimod-loaded ethosomes. The results provide a strong foundation for future formulation development, with the potential to enhance the therapeutic efficacy and skin penetration of Imiquimod, offering an improved treatment strategy for skin cancer.

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Received on 09.12.2024      Revised on 19.02.2025

Accepted on 22.03.2025      Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):426-431.

DOI: 10.52711/0974-360X.2026.00062

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